Fungal diseases have emerged as major life-threatening complications of modern medicine as well as respiratory and systemic disease among both immunocompromised and immunocompetent individuals. The dimorphic fungi in particular can cause disease even among otherwise healthy people. Despite this significant impact on human health, surprisingly few fungal gene products have been identified that promote the virulence of medically important fungi. Yeast cells of the dimorphic fungal pathogen, Histoplasma capsulatum, invade, replicate within, and ultimately kill host macrophages. How Histoplasma succeeds in these tasks is unknown due to significant technical limitations which have hindered progress in understanding Histoplasma pathogenesis at a molecular level. We recently developed powerful molecular tools enabling suppression of Histoplasma gene function thereby providing the means to now functionally define the role of genes in virulence. To address this deficiency in our understanding, this research proposal will identify and define additional fungal virulence determinants among the factors secreted by pathogenic Histoplasma yeast. These extracellular factors are prime candidates for affecting the host macrophages and immune defenses. Taking advantage of recently completed genomic sequence information, extracellular factors will be identified through proteomic-based analyses of proteins secreted by Histoplasma yeast. The genes encoding these factors will then serve as targets for gene silencing by RNA interference or gene deletion methodologies to eliminate their production. This ability to create strains of Histoplasma lacking individual proteins permits functional tests to be performed. Strains will be used to infect human and mouse macrophages in culture to assess intracellular parasitism and will be employed in animal infection models of respiratory histoplasmosis. These experiments are designed to assess the contribution of individual factors in Histoplasma pathogenesis. PUBLIC HEALTH RELEVANCE: Our understanding of the mechanisms by which pathogenic fungi infect and cause disease in human hosts is limited which impairs our options to treat life-threatening fungal disease. This proposal will identify molecules released by fungal cells which allow the pathogen to overcome host immune defenses. Improved understanding of the virulence mechanisms employed by pathogenic fungi, specifically the identification of factors that enable them to cause disease, will facilitate the rational design of better antibiotics and regimens to treat clinical fungal infections.